PROJECT SUMMARY Synaptic transmission relies critically on the rapid uptake of emptied exocytic vesicle membrane remnants from the presynaptic plasma membrane via the coupled and compensatory mechanisms of endocytosis catalyzed by the large GTPase dynamin. Defects in synaptic vesicle recycling have been implicated in various neurological disorders including Epilepsy, Down?s syndrome, Alzheimer?s, Parkinson?s and Huntington?s diseases. Emerging evidence indicates that in addition to dynamin?s better-characterized helical polymerization and mechanoenzymatic membrane constriction activities, a third distinct activity involving the alternative tilting or orientation of its pleckstrin homology (PH) domain at the membrane surface governs synaptic vesicle scission. The mechanisms remain largely uncharacterized. Disease-causing mutations in dynamin, which precipitate centronuclear myopathy (CNM) and Charcot-Marie-Tooth (CMT) disease, map largely to the PH domain or to its various intermolecular interfaces. Although this underscores the importance of the PH domain in dynamin function, it is unclear how these mutations specifically influence PH domain interactions or conformational behavior at the membrane surface. It is our long-term goal to understand the various molecular mechanisms at play in dynamin-mediated endocytic vesicle scission. In this proposal, we seek to address several unknown or unresolved fundamental issues concerning the role of the PH domain in dynamin function, both in solution and on membranes. These include: 1) the regulatory mechanisms and conformational rearrangements that underlie the transition of dynamin from stable, self-limited, cytosolic tetramers to dynamic, self-assembled, membrane- bound helical polymers, 2) the conformational coupling of dynamin PH domain-membrane insertion and alternate orientations to helical self-assembly and the coordination of assembly-dependent GTPase activity, and 3) the molecular nature and structural basis of alternate PH domain orientations on the membrane surface. To address these, we will use a powerful combination of multiple independent fluorescence spectroscopic techniques including Frster resonance energy transfer (FRET), fluorescence lifetime analysis, quenching and stopped-flow kinetic measurements, coupled to sophisticated NMR spectroscopic measurements of the dynamin PH domain on various biomimetic lipid templates. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the mechanisms of dynamin function that underlie rapid synaptic vesicle scission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate synaptic vesicle endocytosis under various disease states.